EP3992209A1 - Cldn18.2-antikörper und verwendung davon - Google Patents

Cldn18.2-antikörper und verwendung davon Download PDF

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EP3992209A1
EP3992209A1 EP20840176.0A EP20840176A EP3992209A1 EP 3992209 A1 EP3992209 A1 EP 3992209A1 EP 20840176 A EP20840176 A EP 20840176A EP 3992209 A1 EP3992209 A1 EP 3992209A1
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Prior art keywords
antibody
seq
antigen
binding fragment
amino acid
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EP3992209A4 (de
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Zhaoyu JIN
Yun Li
Feng Li
Naifan HUO
Xiumei JIN
Li Ren
Zhexian YAN
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Futuregen Biopharmaceutical Beijing Co Ltd
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Futuregen Biopharmaceutical Beijing Co Ltd
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Definitions

  • the present disclosure relates to the field of immunotherapy. Specifically, the present disclosure relates to antibodies that bind to CLDN18.2 and defucosylated forms thereof, as well as uses of the antibodies in the treatment of diseases, especially in cancers.
  • Upper gastrointestinal tumors including gastric cancer and esophageal cancer, are common malignant tumors with poor prognosis worldwide.
  • Gastric cancer particularly has a high occurrence in China, and nearly 42% of the new cases of gastric cancer in the world occurred in China. Due to the lack of early diagnosis, about 80% of gastric cancer patients are already at an advanced stage when they are diagnosed. As gastric cancer is not sensitive to conventional chemotherapeutic drugs, the five-year survival rate of patients with advanced gastric cancer is extremely low, and it has become the third leading cause of death in China. Therefore, the researches in recent years were dedicated to seeking for targeting therapies specific for gastric cancer.
  • Claudin family proteins are the main components of the tight junction structure that is widely distributed in epithelial cells. Similar to the structures of other proteins of Claudin family, CLDN18.2 is a membrane protein with a molecular weight of about 27.8 kd, which has four transmembrane regions and two relatively short extracellular regions (EC1 and EC2). Unlike other proteins of Claudin family, in normal tissue, CLDN18.2 is very specifically expressed only in the highly differentiated epithelial cells of the stomach (Tureci O et al ., Gene, 2011). In tumors derived from gastric epithelial cells, a high proportion of tumor cells express CLDN18.2 on the surface.
  • CLDN18.2 In the lymph nodes and other tissue metastases from gastric cancer cells, high levels of CLDN18.2 expression can also be detected (Woll S. et al ., Int. J. Cancer, 2013). In addition to gastric cancer, CLDN18.2 is also expressed in a high proportion of tumor cells from cholangiocarcinoma, esophageal cancer, pancreatic cancer and the like. As a specific surface marker, CLDN18.2 has become a potential target for cancer treatment. The development of highly efficient antibody drugs against CLDN18.2 will provide the possibility for the treatment of a variety of cancers with great application potential and market value.
  • ADCC effect antibody dependent cell mediated cytotoxicity
  • Fab antigen recognition segments
  • Fc antibody constant region
  • FcyR FC receptors
  • the strength of an antibody binding to an antigen, the abundance of antigen expression and the strength of binding of antibody's FC region to FC receptors on the surface of effector cells can affect the intensity of the ADCC effect and thus the therapeutic effect against cancers (Jefferis and Lund, 2002).
  • the present disclosure provides novel anti-CLDN18.2 antibodies.
  • the above antibodies can bind to CLDN18.2 with high affinity and specificity, and mediate the killing of CLDN18.2-expressing target cells (e.g., tumor cells) by effector cells.
  • defucosylated forms of the above antibodies was generated, which can better bind to Fc ⁇ RIIa and induce ADCC effect than unmodified antibodies.
  • the present disclosure relates to an antibody that binds to CLDN18.2 or an antigen-binding fragment thereof, wherein the antibody has the following heavy chain CDRs (CDRHs) and light chain CDRs (CDRLs):
  • CDRHs heavy chain CDRs
  • CDRLs light chain CDRs
  • the antibody may have a heavy chain constant region sequence as shown in SEQ ID NO: 51 and/or a light chain constant region sequence as shown in SEQ ID NO: 53.
  • the antibody has an Fc region.
  • the antibody may have a glycosyl structure modification at Asn297, wherein the numbering is according to the EU numbering system.
  • the ratio of the glycosyl structure having fucose in the glycosyl structure is 50% or less. In some embodiments, the ratio of the glycosyl structure having fucose in the glycosyl structure is 30% or less, for example 20% or less, 10% or less, 5% or less, 2% or less, or 1% or less.
  • the ratio of the glycosyl structure having fucose in the antibody is 0%-1%.
  • the antibody is produced by a cell with Fut8 gene knockout.
  • the cell may be selected from a CHO cell and a HEK293 cell.
  • the antibody has an increased Fc ⁇ RIIIa binding activity as compared to an antibody produced in a cell without Fut8 gene knockout. In some embodiments, the antibody has an increased ADCC activity as compared to an antibody produced in a cell without Fut8 gene knockout.
  • the antibody may be a monoclonal antibody. In other embodiments, the antibody may be a bispecific antibody or a multispecific antibody.
  • the antibody may be selected from a group consisting of IgG, IgA, IgM, IgE, and IgD isotypes. In some embodiments, the antibody may be selected from a group consisting of IgG1, IgG2, IgG3, and IgG4 subclasses.
  • the antigen-binding fragment may be selected from a group consisting of Fab fragment, Fab' fragment, F(ab') 2 fragment, Fd fragment, Fd' fragment, Fv fragment, scFv fragment, ds-scFv fragment, dAb fragment, single chain fragment, diabody and linear antibody.
  • the present disclosure relates to a nucleic acid molecule, wherein the nucleic acid molecule comprises a nucleotide sequence encoding the antibody or antigen-binding fragment thereof of the present disclosure.
  • the present disclosure relates to a vector comprising a nucleic acid molecule of the present disclosure.
  • the present disclosure relates to a host cell comprising the nucleic acid molecule or vector of the present disclosure.
  • the present disclosure relates to a conjugate comprising any antibody or antigen-binding fragment thereof of the present disclosure conjugated to a therapeutic agent, a diagnostic agent or an imaging agent.
  • the present disclosure relates to a composition
  • a composition comprising the antibody or antigen-binding fragment thereof or the conjugate of the present disclosure, and one or more pharmaceutically acceptable carriers, excipients, and/or diluents.
  • the composition further comprises one or more additional therapeutic agents.
  • the therapeutic agents may be selected from a group consisting of antibody, chemotherapeutic, and small molecule drug.
  • the chemotherapeutic may be selected from one or more of Epirubicin, Oxaliplatin, and 5-Fluorouracil (5-FU).
  • the present disclosure relates to a method of treating a disease associated with the expression of CLDN18.2 in a subject, the method comprises a step of administering to the subject the antibody or antigen-binding fragment thereof, or the conjugate, or the composition of the present disclosure.
  • the disease is a cancer.
  • the cancer may be selected from a group consisting of gastric cancer, cholangiocarcinoma, esophageal cancer, and pancreatic cancer.
  • the method further comprises a step of administering to the subject one or more additional therapies, for example, a cancer therapy.
  • the additional therapy is selected from a group consisting of chemotherapy, radiation therapy, immunotherapy, and surgery.
  • the immunotherapy is selected from a group consisting of therapy targeting an immune checkpoint molecule, CAR-T cell therapy, and CAR-NK cell therapy.
  • the chemotherapy is selected from a combined chemotherapy regimen comprising Epirubicin, Oxaliplatin, and 5-fluorouracil.
  • the present disclosure relates to use of the antibody or antigen-binding fragment thereof, the conjugate, or the composition of the present disclosure in treating a disease associated with the expression of CLDN18.2 in a subject.
  • the present disclosure relates to use of the antibody or antigen-binding fragment thereof, the conjugate, or the composition of the present disclosure in the manufacture of a medicament for treating a disease associated with the expression of CLDN18.2 in a subject.
  • the disease is a cancer.
  • the cancer may be selected from a group consisting of gastric cancer, cholangiocarcinoma, esophageal cancer, and pancreatic cancer.
  • the present disclosure relates to a polypeptide having an amino acid sequence selected from SEQ ID NOs: 1, 6, 11, 16, 21, 26, 31, 36, 41, 46, 55, and 60.
  • the present disclosure relates to a nucleic acid molecule having a nucleotide sequence selected from SEQ ID NOs: 2, 7, 12, 17, 22, 27, 32, 37, 42, 47, 56, and 61.
  • the present disclosure also relates to a vector comprising above nucleic acid molecule.
  • Antibody Ab; immunoglobulin: Ig;
  • the present disclosure relates to an antibody that binds to CLDN18.2 or an antigen-binding fragment thereof, wherein the antibody has the following heavy chain CDRs (CDRHs) and light chain CDRs (CDRLs):
  • CDRHs heavy chain CDRs
  • CDRLs light chain CDRs
  • binding refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and the antigen against which it is directed.
  • an antibody that specifically binds to a certain antigen refers to an antibody binding to the antigen with an affinity (K D ) of less than about 10 -5 M, for example less than about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, or 10 -10 M or less.
  • K D refers to the dissociation equilibrium constant of a particular antibody-antigen interaction and is used to describe the binding affinity between an antibody and an antigen. The smaller the dissociation equilibrium constant, the tighter the antibody-antigen binding, and the higher the affinity between the antibody and the antigen.
  • the term “antibody” refers to an immunoglobulin molecule that comprises at least one antigen recognition site and is able to specifically bind to an antigen.
  • antigen refers to a substance, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, hapten, or a combination thereof, that induces an immune response in the body and binds specifically to an antibody.
  • the binding of an antibody and an antigen is mediated by the interaction formed between the two, including hydrogen bond, van der Waals force, ionic bond, and hydrophobic bond.
  • the region of an antigen surface to which an antibody binds is an "antigenic determinant " or "epitope", and in general, each antigen may have multiple epitopes.
  • epitope may be formed by contiguous amino acids or non-contiguous amino acids that are brought together by the folding of a protein.
  • An Epitope typically comprises at least 3, more often at least 5, about 9, or about 8-10 amino acids in a unique spatial conformation.
  • An “epitope” includes a structural unit normally bound by an immunoglobulin VH/VL pair.
  • An epitope defines the smallest binding site of an antibody and thus represents a target specific to an antibody or antigen-binding fragment thereof.
  • antibody as used in this disclosure is understood in its broadest meaning, and includes monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, antibody fragments, multi-specific antibodies comprising at least two different antigen-binding structural domains (e.g., bispecific antibodies).
  • Antibody also includes mouse-derived antibodies, humanized antibodies, chimeric antibodies, human antibodies, and antibodies from other sources. Antibody may contain additional alterations, such as unnatural amino acids, Fc effector function mutations, and glycosylation site mutations.
  • Antibody may also include post-translationally modified antibodies, fusion proteins comprising the antigenic determinant cluster of antibodies, and immunoglobulin molecules comprising any other modifications to the antigen recognition site, provided that these antibodies exhibit the desired biological activity. In other words, include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules that contain at least one antigen-binding domain.
  • variable region refers to paired light and heavy chain domain portions that directly participate in the binding of an antibody and an antigen.
  • VH and VL region consists of three highly variable regions or complementary determining regions (CDRs) and four framework regions (FRs) arranged from N-terminal to C-terminal in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • CDR refers to the complementary determining region within the variable sequence of an antibody.
  • CDR1, CDR2, and CDR3 are three CDRs in each variable region of the heavy chain and light chain.
  • CDR1, CDR2, and CDR3 The exact boundaries of these CDRs are defined differently according to different systems.
  • the system described by Kabat et al. Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987) and (1991 ) provides not only an explicit residue numbering system applicable to antibody variable regions, but also residue boundaries that define three CDRs. These CDRs may be called Kabat CDRs.
  • Each complementary determining region can comprise the amino acid residues of the "complementary determining region" defined by Kabat. Chothia et al. ( Chothia & Lesk, J. Mol. Biol, 196:901-917 (1987 ) and Chothia et al., Nature 342:877-883 (1989 )) found that certain subparts within the Kabat CDRs adopt an almost identical peptide backbone conformation, despite the diversity at the amino acid sequence level. These subparts are called L1, L2 and L3 or HI, H2 and H3, respectively, where "L” and “H” denote the light and heavy chain regions, respectively. These regions can be called Chothia CDRs, which have overlapping boundaries with Kabat CDRs.
  • the CDRH1, CDRH2, and CDRH3 in a VH as shown in SEQ ID NO: 1 have amino acid sequences of SEQ ID NO: 3 (SSWLI), SEQ ID NO: 4 (TIVPSDSYTNYNQKFKD) and SEQ ID NO: 5 (FRTGNSFDY), respectively
  • the CDRL1, CDRL2, and CDRL3 in a VL as shown in SEQ ID NO: 6 have amino acid sequences of SEQ ID NO: 8 (KSSQSVLNSGNQKNYLT), SEQ ID NO: 9 (WAVARQS) and SEQ ID NO: 10 (QNSIAYPFT), respectively;
  • the antibody has the following CDRHs and CDRLs:
  • the antibody has the following VH and VL:
  • the antibody has the following VH and VL:
  • the amino acid modification does not change the CDR sequences of the antibody, i.e., the amino acid modification is performed in a framework region (FR) of a variable region.
  • FR framework region
  • the one or more amino acid modifications refer to 1-10 amino acid modifications or 1-5 amino acid modifications, for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid modifications.
  • amino acid modifications are selected from substitution, deletion, addition and/or insertion of amino acid residues.
  • said amino acid modifications are amino acid substitutions, for example conservative substitutions.
  • the antibody has the following VH and VL:
  • sequence identity As understood by those skilled in the art, the correlation between two amino acid sequences or between two nucleotide sequences can be described by the parameter "sequence identity".
  • the percentage of sequence identity between two sequences can be determined, for example, by using a mathematical algorithm.
  • the percentage of sequence identity between two sequences can be determined, for example, by using a mathematical algorithm.
  • Non-limiting examples of such mathematical algorithms include the algorithm of Myers and Miller (1988) CABIOS 4:11-17 , the local homology algorithm of Smith et al., (1981) Adv. Appl. Math. 2:482 , the homology comparison algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443- 453 , the method for searching for homology of Pearson and Lipman (1988) Proc.
  • sequence comparisons i.e., alignments
  • the programs can be properly executed by a computer. Examples of such programs include, but are not limited to, CLUSTAL of the PC/Gene program, the ALIGN program (Version 2.0), and GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics software package.
  • the alignment using these programs can be implemented, for example, by using initial parameters.
  • the antibody may have a heavy chain constant region sequence as shown in SEQ ID NO: 51 and/or a light chain constant region sequence as shown in SEQ ID NO: 53.
  • the antibody has an Fc region.
  • the antibody has a glycosyl structure modification at Asn297, wherein the numbering is according to the Eu numbering system.
  • “Asn297” according to the present disclosure means asparagine located at position 297 in the Fc region of an antibody according to the Eu numbering system. Asn297 may also be located a few amino acids upstream or downstream of position 297 based on subtle sequence changes of the specific antibody.
  • the term "Fc region of an antibody” or “human immunoglobulin Fc region” comprises the constant region polypeptide of an antibody other than the heavy chain constant region 1 (CH1), for example, two constant region domains (CH2 and CH3) at the carboxyl terminus of the heavy chain constant regions of human immunoglobulins IgA, IgD, IgG, and three constant region domains (CH2, CH3 and CH4) at the carboxyl terminus of the heavy chain constant regions of human immunoglobulins IgE and IgM, and also includes the flexible hinge regions at the amino terminus of these domains.
  • the heavy chain Fc region of human IgG is typically defined as comprising the residues starting from A231 to its carboxy terminus.
  • the Fc region of immunoglobulin is the functional domain which exerts the immune effect of an antibody.
  • the Fc of an IgG antibody can interact with a variety of receptors, the most important of which is the Fc ⁇ receptor (FcyR) family.
  • FcyR Fc ⁇ receptor
  • a major mechanism of action of therapeutic monoclonal antibodies to kill tumor cells is the ADCC effect (antibody dependent cell mediated cytotoxicity).
  • ADCC effect antibody dependent cell mediated cytotoxicity
  • the Fc region of the antibody binds to killer cells expressing FcyR and activate the cell killing activity of the effector cells, thus secreting cytotoxic mediators including granzymes, perforins, etc., which ultimately lead to the lysis and destruction of target cells (e.g. tumor cells).
  • Fc can also bind to the complement protein C1q and produce a CDC effect (complement dependent cytotoxicity, CDC).
  • the level and form of glycosylation modification in the Fc region of an antibody can affect the binding ability of the Fc region to its receptor FcyR, thereby affecting the strength of the ADCC effect of the antibody.
  • the glycosylation modification of the Fc region of an antibody usually refers to the glycosylation modification at Asn297.
  • the level of fucosylation of the antibody facilitates the increased binding of the Fc region to Fc ⁇ RIIIa, thereby enhancing the ADCC effect of the antibody.
  • altering the metabolic pathway of glycosylation in antibody-expressing cells can effectively alter the level of fucosylation in expressed antibodies, thereby modulating the antibody-mediated ADCC effect ( Jefferis R. 2009, NAT. REV. Drug. DISC ).
  • the antibody has a reduced level of fucosylation.
  • the ratio of the glycosyl structure having fucose in the glycosyl structure at Asn297 is 60% or less, e.g., 50% or less, e.g., 40% or less, 30% or less, 20% or less, 10% or less, 5% or less, 2% or less, or 1% or less.
  • the ratio of the glycosyl structure having fucose is 0%-10%, e.g., 0%-5%, 0%-2%, or 0%-1%.
  • the ratio of the glycosyl structure having fucose is 0.1% or less. In other embodiments, there is no detectable fucose in the glycosyl structure at Asn297.
  • Such defucosylated antibodies can be generated using techniques known to those skilled in the art.
  • the antibodies can be expressed in cells that are defective of or lack of the ability to fucosylation.
  • cell lines with Fut8 gene knockout can be used to produce antibodies with reduced levels of fucosylation.
  • antibody or antigen-binding fragment having reduced or no fucose content can be produced by, for example: (i) culturing cells under conditions that prevent or reduce fucosylation; (ii) removing fucose after translation (e.g., with fucosidase); (iii) adding desired carbohydrates after translation, e.g., after recombinant expression of non-glycosylated glycoproteins; or (iv) purifying glycoproteins to select for the antibody or antigen-binding fragment thereof that is not fucosylated.
  • the antibody is produced by a cell with Fut8 gene knockout to obtain the antibody with reduced level of fucosylation.
  • the cell is Chinese Hamster Ovary (CHO) cell, such as CHO-K1 cell, CHOS cell, or other CHO-derived cells.
  • the cell is CHO cell with Fut8 gene knockout.
  • the cell is human embryonic kidney (HEK) 293 cell, such as HEK293, HEK293A, HEK293T, HEK293F, or other HEK293-derived cells.
  • HEK 293 cell with Fut8 gene knockout.
  • the antibody is produced by a cell with Fut8 gene knockout, wherein the antibody has an increased Fc ⁇ RIIIa binding activity, as compared to an antibody produced in a cell without Fut8 gene knockout, e.g., a control antibody with the identical sequence.
  • an antibody produced by a cell with Fut8 gene knockout has an increased ability to induce activity as compared to an antibody produced in a cell without Fut8 gene knockout, e.g., a control antibody with the identical sequence.
  • an antibody produced by a cell with Fut8 gene knockout has at least 2-fold, e.g., at least 5-fold or at least 10-fold, e.g., 10 to 20-fold, increased EC50 values for inducing Fc ⁇ RIIIa activity, as compared to an antibody produced in a cell without Fut8 gene knockout.
  • EC50 refers to the concentration of an antibody inducing 50% of the maximum effect.
  • the antibody is produced by a cell with Fut8 gene knockout, and wherein the antibody has an increased ADCC activity, as compared to an antibody produced in a cell without Fut8 gene knockout, e.g., a control antibody with the identical sequence.
  • an antibody produced by a cell with Fut8 gene knockout has at least 2-fold, e.g., at least 5-fold or at least 10-fold, e.g., 10 to 20-fold increased ADCC activity, as compared to an antibody produced in a cell without Fut8 gene knockout.
  • the antibody is a monoclonal antibody.
  • the term “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous antibody population, i.e., each antibody constituting the population is identical, except for possible naturally occurring mutations that may exist in small amounts.
  • a monoclonal antibody is highly specific and is directed against a single antigen.
  • each antibody in a monoclonal preparation is directed against the same single determinant on an antigen.
  • the term “monoclonal antibody” is not limited to an antibody produced by hybridoma technology, and the modifier "monoclonal antibody” should not be interpreted as requiring the production of an antibody by any particular method.
  • the antibody is a bispecific antibody or a multispecific antibody.
  • the bispecific or multispecific antibody has a first antigen-binding region that binds to an epitope on CLDN 18.2, and a second antigen-binding region that binds to another antigenic epitope, wherein the first antigen-binding region has the CDRH1, CDRH2 and CDRH3 as well as CDRL1, CDRL2 and CDRL3, or VH and VL sequences of the antibody or antigen-binding fragment thereof as described in the present disclosure, and the second antigen-binding region binds to a different antigenic epitope than the first antigen-binding region.
  • the second antigen-binding region binds to another antigen-binding epitope on the CLDN18.2 molecule. In other embodiments, the second antigen-binding region binds to another antigen. In some embodiments, said another antigen is selected from tumor-associated antigens and immune checkpoint molecules.
  • tumor-associated antigen refers to antigen that is differentially expressed by cancer cells and therefore can be utilized to target cancer cells.
  • Tumor-associated antigen is an antigen that can potentially stimulate a significant tumor-specific immune response.
  • Some of these antigens are encoded by normal cells, but not necessarily expressed by normal cells. These antigens can be characterized as those that are usually silent (i.e., not expressed) in normal cells, those that are expressed only at certain stages of differentiation, and those that are expressed over time, such as embryonic and fetal antigens.
  • cancer antigens are encoded by mutant cytogenes such as oncogenes (e.g., activated ras oncogenes), suppressor genes (e.g., mutant p53), and fusion proteins produced by internal deletions or chromosomal translocations.
  • mutant cytogenes such as oncogenes (e.g., activated ras oncogenes), suppressor genes (e.g., mutant p53), and fusion proteins produced by internal deletions or chromosomal translocations.
  • cancer antigens can be encoded by viral genes, such as those carried on RNA and DNA tumor viruses. Many other tumor-associated antigens and antibodies against them are known and/or commercialized, and can also be generated by those skilled in the art.
  • Immune checkpoint protein receptors and their ligands mediate the suppression of T cell-mediated cytotoxicity, and are typically expressed by tumors or on nonreactive T cells in the tumor microenvironment, and allow tumors to evade immune attack.
  • Inhibitors of the activity of immunosuppressive checkpoint protein receptors and their ligands can overcome the immunosuppressive tumor environment to allow cytotoxic T-cell attack of tumors.
  • immune checkpoint proteins include, but are not limited to, PD-1, PD-L1, PD-L2, CTLA4, OX40, LAG3, TIM3, TIGIT, and CD103.
  • immune checkpoint modulators can include, for example, antibodies targeting checkpoint proteins, aptamers, small molecules, and soluble forms of checkpoint receptor proteins.
  • Antibodies specific for PD-1, PD-L2, CTLA4, OX40, LAG3, TIM3, TIGIT, and CD103 are known and/or commerciallized, and can also be generated by those skilled in the art.
  • immunoglobulins can be divided into 5 classes (isotypes): IgA, IgD, IgE, IgG and IgM, which can be further divided into different subclasses, such as IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, etc.
  • amino acid sequence of light chain light chains can be classified as ⁇ chain or ⁇ chain.
  • the antibody of the present disclosure can be any of the above-mentioned classes or subclasses.
  • the antibody of the present disclosure is selected from a group consisting of IgG, IgA, IgM, IgE, and IgD isotypes. In some embodiments, the antibody of the present disclosure is IgG, for example selected from a group consisting of IgG1, IgG2, IgG3, and IgG4 subclasses.
  • the term "antigen-binding fragment” includes, but is not limited to: Fab fragment having VL, CL, VH and CHI domains; Fab' fragment, which is a Fab fragment with one or more cysteine residues at the C-terminus of the CHI domain; Fd fragment, which has VH and CHI domains; Fd' fragment, which has VH and CHI domains and one or more cysteine residues at the C-terminus of the CHI domain; Fv fragment and scFv, which have the VL and VH domains of a single arm of an antibody; dAb fragment, which consists of either a VH or VL domain; isolated CDR region; F(ab') 2 fragment, which is a bivalent fragment comprising two Fab' fragments linked by a disulfide bridge at the hinge region; single-chain antibody molecule ( e.g., single chain Fv; scFv); "diabody" having two antigen binding sites, which comprises a heavy-chain molecule
  • the antigen-binding fragment is selected from a group consisting of Fab fragment, Fab' fragment, F(ab') 2 fragment, Fd fragment, Fd' fragment, Fv fragment, scFv fragment, ds-scFv fragment, dAb fragment, single chain fragment, diabody and linear antibody.
  • the present disclosure relates to a nucleic acid molecule comprising a nucleotide sequence encoding the antibody or antigen-binding fragment thereof of the present disclosure.
  • the present disclosure also relates to a vector comprising the nucleic acid molecule of the present disclosure.
  • vector refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted.
  • the vector When the vector enables the expression of the proteins encoded by the inserted polynucleotide, the vector is called an expression vector.
  • the vector can be introduced into a host cell by methods such as transformation, transduction, or transfection, and subsequently a genetic material element it carries can be expressed in the host cell.
  • the vector is recognized by those skilled in the art, including but not limited to, (1) plasmids; (2) phage particles; (3) Coase plasmids; (4) artificial chromosomes, such as yeast artificial chromosomes, bacterial artificial chromosomes or PI-derived artificial chromosomes; (5) phages such as ⁇ phages or M13 phages and (6) animal viruses, such as retroviruses, adenoviruses, adeno-associated viruses, sporoviruses, poxviruses, and baculoviruses.
  • a vector may contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription start sequences, enhancer sequences, selection elements, and reporter genes; in addition, the vector may contain replication initiation sites.
  • the present disclosure relates to a host cell comprising the nucleic acid molecule or vector of the present disclosure.
  • the host cell is CHO cell, e.g., CHO-K1 cell, CHOS cell, or other CHO-derived cells.
  • the host cell is HEK 293 cell, for example HEK293, HEK293A, HEK293T, HEK293F, or other HEK293-derived cell.
  • the present disclosure relates to a conjugate comprising the antibody or antigen-binding portion thereof of the present disclosure conjugated to a therapeutic agent, a diagnostic agent, or an imaging agent.
  • the therapeutic agent may be selected from cytotoxins and radioisotopes.
  • the diagnostic agent or imaging agent may be selected from a group consisting of fluorescent markers, luminescent substances, chromogenic substances, and enzymes.
  • the present disclosure relates to a composition
  • a composition comprising the antibody or antigen-binding fragment thereof or the conjugate of the present disclosure, and one or more pharmaceutically acceptable carriers, excipients, and/or diluents.
  • phrases “pharmaceutically acceptable” refers to those compounds, materials, compositions, and/or dosage forms that are suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic response, or other problems or complications, within the scope of reasonable medical judgment, commensurate with a reasonable benefit/risk ratio.
  • pharmaceutically acceptable carriers, excipients and/or diluents refers to pharmaceutically acceptable materials, compositions or vehicles, such as liquid or solid fillers, diluents, excipients, solvents, medium, encapsulating materials, manufacturing aids or solvent encapsulating materials, which are involved in maintaining the stability, solubility, or activity of the antibody or antigen-binding fragment thereof of the present disclosure.
  • composition of the present disclosure can be formulated for administration to a subject in a solid, liquid, or gel form.
  • the composition of the present disclosure can be formulated for parenteral administration, e.g., by subcutaneous, intramuscular, intravenous or epidural injection, as, e.g., a sterile solution or suspension or a sustained release formulation.
  • the composition further comprises one or more additional therapeutic agents.
  • the additional therapeutic agent is selected from a group consisting of antibody, chemotherapeutic, and small molecule drug.
  • the therapeutic agent targets a tumor-associated antigen, e.g., a tumor-associated antigen as described above.
  • the therapeutic agent targets an immune checkpoint molecule, e.g., an immune checkpoint molecule as described above.
  • chemotherapeutic refers to any chemical agent that has therapeutic usefulness in the treatment of diseases characterized by abnormal cell growth.
  • the chemotherapeutic includes both chemical and biological agents. These agents function to inhibit the cellular activity upon which cancer cells depend to achieve sustained survival.
  • the categories of chemotherapeutic agents include alkylating/alkaloid agents, antimetabolites, hormones or hormone analogs, and various anti-neoplastic drugs.
  • the additional therapeutic agent is a chemotherapeutic, wherein the chemotherapeutic may be selected from one or more of Epirubicin, Oxaliplatin, and 5-fluorouracil (5-FU).
  • the present disclosure relates to a method of treating a disease associated with the expression of CLDN18.2 in a subject, comprising a step of administering to the subject the antibody or antigen-binding fragment thereof, conjugate or composition of the present disclosure.
  • treatment refers to a therapeutic treatment in which the purpose is to reverse, reduce, improve, inhibit, slow down, or stop the progression or severity of a symptom associated with a disease or condition.
  • treatment includes reducing or alleviating at least one side effect or symptom of a disease or condition.
  • a treatment is generally “effective” if it reduces one or more symptoms or clinical markers.
  • a treatment is “effective” if the progression of a disease is reduced or stopped, that is, the "treatment” includes not only an improvement in a symptom but also a cessation, or at least slowing down of the progression or worsening of a symptom that would be expected in the absence of treatment.
  • Beneficial or desired clinical outcomes include, but are not limited to, alleviation of one or more symptoms, reduction in the extent of a disease, stabilization (i.e., no worsening) of a disease state, delay or slowing down of disease progression, improvement or alleviation of a disease state, and remission (whether partial or all), whether detectable or undetectable.
  • the terms “subject”, “patient”, and “individual” are used interchangeably herein, and refer to an animal, e.g., a human.
  • the term subject also includes "non-human mammal”, for example, rat, mouse, rabbit, sheep, cat, dog, cattle, pig, and non-human primate.
  • the subject is a human subject.
  • the disease is a cancer.
  • the cancer include, but are not limited to: basal cell carcinoma, cholangiocarcinoma; bladder cancer; bone cancer; breast cancer; peritoneal cancer; cervical cancer; bile duct cancer; choriocarcinoma; colon and rectal cancer; connective tissue cancer; digestive system cancer; endometrial cancer; esophageal cancer; eye cancer; head and neck cancer; stomach cancer; glioblastoma; liver cancer; kidney cancer; laryngeal cancer; leukemia; liver cancer; lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and squamous cell lung cancer); lymphoma, including Hodgkin's lymphoma and non-Hodgkin's lymphoma; melanoma; myeloma; neuroblastoma; oral cancer; ovarian cancer; pancreatic cancer; prostate cancer; retino
  • the method further comprises a step of administering one or more additional therapies.
  • the therapy is selected from a group consisting of chemotherapy, radiotherapy, immunotherapy, and surgical therapy.
  • the immunotherapy is selected from a group consisting of a therapy targeting an immune checkpoint molecule, a CAR-T cell therapy, and a CAR-NK cell therapy.
  • the immune checkpoint molecule may be selected from a group consisting of PD-1, PD-L1, PD-L2, CTLA4, OX40, LAG3, TIM3, TIGIT, and CD103.
  • the chemotherapy is selected from a combined chemotherapy regimen including Epirubicin, Oxaliplatin, and 5-fluorouracil.
  • the present disclosure relates to use of the antibody or antigen-binding fragment thereof, the conjugate, or the composition of the present disclosure in treating a disease associated with the expression of CLDN18.2 in a subject. In another aspect, the present disclosure relates to use of the antibody or antigen-binding fragment thereof, the conjugate, or the composition of the present disclosure in the manufacture of a medicament for treating a disease associated with the expression of CLDN18.2 in a subject.
  • the disease is a cancer, for example, the cancer types described above.
  • the cancer is selected from a group consisting of gastric cancer, cholangiocarcinoma, esophageal cancer, and pancreatic cancer.
  • the subject is a human.
  • the present disclosure relates to a polypeptide having an amino acid sequence selected from SEQ ID NOs: 1, 6, 11, 16, 21, 26, 31, 36, 41, 46, 55, and 60.
  • the present disclosure relates to a nucleic acid molecule having a nucleotide sequence selected from SEQ ID NOs: 2, 7, 12, 17, 22, 27, 32, 37, 42, 47, 56, and 61.
  • the present disclosure also relates to a vector comprising above-mentioned nucleic acid molecule.
  • antibody drugs is currently mainly achieved by humanizing antibodies derived from other species or directly screening fully human antibodies through transgenic animals and in vitro screening techniques, which further reduces the immunogenicity of antibodies in humans, reduces the corresponding side effects, and improves drugability, which is an important trend in antibody drug development.
  • Phage display antibody library technology is one of the main methods to obtain fully human antibodies.
  • Phage display technology uses molecular biology methods to insert exogenous gene fragments into genes of phage-specific proteins such as gIII, expresses the exogenous gene-encoded protein or peptide by the phage, and presents them on the phage surface while maintaining the relative spatial structure and biological activity of the recombinant fusion proteins.
  • the constructed diverse phage library is co-incubated with the target protein, and non-target protein-binding phage strains are removed by biopanning. With sufficient phage library capacity, phage clones with high affinity and high specificity can be obtained through multiple rounds of collection, amplification and enrichment. Gene sequencing can be used to identify the protein sequences encoded by these phage clones for further researches.
  • Phage antibody library is a diverse phage library formed by phage display of antibody genes.
  • the quality of the phage antibody library is mainly determined by its capacity and diversity.
  • the phage antibody library needs to be as large as possible while maintaining diversity.
  • the number of antibody fragments displayed on the phage surface represents the size of the library capacity, while the diversity of displayed fragments represents the antibody library diversity. Theoretically, the larger the library capacity of the phage antibody library, the higher the affinity of the antibody obtained by screening.
  • RNA samples of total RNAs of mononuclear cells were obtained, collected and extracted from the peripheral blood and spleen of healthy adult individuals and from the umbilical cord blood of newborns.
  • the RNA was reverse-transcribed to synthesize single-stranded cDNA, and then the VH, V ⁇ and V ⁇ genes were amplified using variable region primers for different subgroups of antibodies, respectively.
  • the amplification products were mixed in a certain ratio, and the heavy and light chain genes were respectively linked by PCR to form single chain antibodies (scFv), and cloned into phage plasmids by dual enzyme cleavage.
  • SS320 E. coli competent cells were transformed by electroporation using the phage plasmids carrying the scFv genes. After SS320 proliferated to log phase, the helper phage was added for infection.
  • a natural antibody phage library with a library capacity of 1.2 ⁇ 10 12 and a positive rate of 88% was obtained.
  • the gene family of the antibody library was found to be close to the natural distribution by sequencing randomly selected clones, and the number of amino acids in the CDR3 region was 3-20. No antibody repeats were found during the sequencing, and 88% of the gene sequences had the correct reading frames. The above results indicated that the phage antibody library had good diversity and had a high effective library capacity.
  • a CHO cell line stably expressing CLDN18.2 (CHO-CLDN18.2) was prepared for use in cell-based phage display screening.
  • the specific experimental procedure was as follows. The cDNA sequence of human CLDN18.2 was cloned into a pCDH lentiviral vector, and then co-transfected with a lentiviral packaging vector into 293t cells. After 48 or 72 hours of incubation, the cell supernatants enriched with lentiviral particles were collected and used for the direct infection with CHO cells, CT26 cells or SNU601 cells.
  • the cells infected with lentivirus were subjected to screening and culture using puromycin, and after 2-3 weeks, the CLDN18.2 expression was tested by flow cytometry using CLDN18.2 specific antibody (IMAB362, Ganymed), the results are shown in Figure 1 .
  • CLDN18.2 specific antibody IMAB362, Ganymed
  • CHO cells named CHO-CLDN18.2 cell line
  • CT26 cells CT26-CLDN18.2
  • SNU601 cells SNU601-CLDN18.2 stably expressing CLDN18.2 were obtained by lentiviral transfection and puromycin screening.
  • the experimental procedure mainly refers to the following literature: Targeting membrane proteins for antibody discovery using phage display Jones ML et al ., Scientific Reports 2016.
  • the process is briefly described as follows.
  • the phage library were incubated with CHO-K1 cells and the supernatant was taken after centrifugation. Then the supernatant was incubated with CHO-CLDN18.2 cells to allow the CLDN18.2 specific phages to bind to the cells. Cell precipitates were collected after centrifugation, and the cells were washed and collected. Then the phage library bound to the cells were eluted using 75 mM sodium citrate buffer. After neutralization of the phage library, the phage library after screening was amplified 100-fold using M13K07-assisted phage, followed by a second and a third round of screening in a similar manner to the first round of screening.
  • the enrichment of phage was monitored by the initial phage amount of each round of screening and the phage titer collected after screening, and was tested by flow cytometry with the phage library and the CHO or CHO-CLDN18.2 stable expression cell lines.
  • the results shown in Figure 2 indicate that the phage display library can specifically bind to CHO-CLDN18.2 starting from the second round of enrichment screening, and its binding intensity increased with the number of screenings.
  • Enriched phage library was obtained after three rounds of screening. After infected by the library, the bacteria were spread on agarose plates for culture. The monoclonal colonies were picked and placed in a 96-well deep-well plate with 2YT medium containing ampicillin and kanamycin, and were cultured with shaking at 37°C to obtain supernatants containing monoclonal phages. The monoclonal phage supernatants were incubated with CHO-CLDN18.2 cells or CT26-CLDN18.2 cells at 4°C for 1 h, followed by washing with PBS (flow cytometry buffer) containing 1 mM EDTA and 0.5% BSA.
  • PBS flow cytometry buffer
  • PE-labeled anti-M13 antibody purchased from Yiqiao Shenzhou was added and was incubated at 4°C for 30 min. Subsequently, the binding of phages to cells was tested by flow cytometry, and the results are shown in Figure 3 .
  • the cDNA sequences of the heavy and light chain variable regions of the monoclonal phages were cloned into pcDNA3.4 vectors (Invitrogen) which already contained the antibody constant region, respectively, and a total of 6 plasmids expressing monoclonal antibody heavy and light chain were obtained.
  • the plasmids were transiently transfected into EXPI-293 cells (Invitrogen) using the PEI method for 7-10 days and centrifuged and supernatants were collected. The supernatants were purified by protein A to obtain purified antibodies.
  • the 6 monoclonal antibodies above were named 18.2-A1 (A1), 18.2-A2 (A2), 18.2-A3 (A3), 18.2-A4 (A4), 18.2-A5 (A5) and 18.2-A6 (A6).
  • the amino acid sequences and the coding sequences of the heavy and light chain variable regions of these antibodies are shown in Tables 1 and 2 below, and the CDR sequences of the antibodies determined according to the Kabat CDR system are shown in Table 3.
  • the sequences of the heavy and light chain constant regions of the recombinant antibodies are shown in Table 4. Table 1.
  • Sequences of heavy and light chain constant regions of recombinant antibodies Sequence of heavy chain constant region SEQ ID NO: 51 Coding sequence of heavy chain constant region SEQ ID NO: 52 Sequence of light chain constant region SEQ ID NO: 53 Coding sequence of light chain constant region SEQ ID NO: 54
  • Example 5 Tests for the binding activities of recombinant monoclonal antibodies
  • Binding activities of the recombinant monoclonal antibodies to CLDN18.2 were determined. Specifically, well-grown cell lines stably expressing CT26-CLDN18.2 were taken. The cells were washed with PBS, mixed with the fold-diluted antibody and incubated at 4°C for 1 hour. The cells were washed once with flow cytometry buffer, PE-labeled anti-human IgG antibody was added, and incubated at 4°C for 30 minutes. The supernatant was removed by centrifugation, and after the cells were washed with flow cytometry buffer, the fluorescence intensity on the surface of the cells was tested by flow cytometry. The relative binding activity of each antibody was calculated using the average fluorescence intensity.
  • 18.2-A1 (A1), 18.2-A2 (A2), 18.2-A3 (A3), 18.2-A4 (A4) 18.2-A5 (A5) and 18.2-A6 (A6) antibodies did not bind to the control CT26 cell line (results are not shown), but were able to bind well to the CT26 cell line expressing CLDN18.2, as shown in Figure 4 .
  • the average fluorescence intensity of CT26-CLDN18.2 gradually decreased with the dilution of the antibody.
  • the relative binding activities (EC50) of the above CLDN18.2 antibodies are also shown in Figure 4 .
  • Human Claudin18 proteins include two homologues, i.e., CLDN18.1 and CLDN18.2, which share exons other than exon 1, and the exon 1 sequences of CLDN18.1 and CLDN18.2 are highly similar with only 8 different amino acids in the first extracellular region.
  • the exon 1 of CLDN18.1 and the exon 1 of CLDN18.2 are specifically expressed in different tissues driven by different promoters. Among them, CLDN18.1 is specifically expressed in lung epithelial cells, while CLDN18.2 is specifically expressed in gastric epithelial cells. To identify the specificity of the screened CLDN18.2 antibodies, binding of the antibodies to CLDN18.1 and CLDN18.2 was tested.
  • the supernatant was removed by centrifugation, wash was performed twice with flow cytometry buffer, and the cells were fixed in a fix solution containing 1% paraformaldehyde and 0.5% triton-X100 and the cell membrane was permeabilized. Subsequently, the cells were incubated with APC-labeled anti-flag antibody for 30 min and tested by flow cytometry.
  • the CLDN18.2 antibodies 18.2-A1, 18.2-A3, 18.2-A4, 18.2-A6 or IMAB362 antibody (as described in Example 2) at a concentration of 10 ⁇ g/ml, blank control (PBS) and isotype control (ISO) were incubated with the cells for 4 h at 4°C or 37°C, respectively. Subsequently, they were washed at 4°C and stained with PE-labeled anti-human IgG antibody. After fixation with 4% formalin, antibody binding on the cell surface was tested by flow cytometry.
  • IMAB362 antibody and 18.2-A1, 18.2-A3, 18.2-A4 and 18.2-A6 antibodies all bound well to SNU601 cells expressing CLDN18.2 under 4°C incubation condition.
  • IMAB362 antibody was endocytosed by the cells and the antibody staining on the cell surface decreased significantly; whereas 18.2-A1, 18.2-A3, 18.2-A4 and 18.2-A6 antibodies did not undergo significant endocytosis and still bound well to the cells under 37°C condition.
  • IMAB362 antibody could induce cellular endocytosis, which may down-regulate the expression level of CLDN18.2 antigen on the cell surface.
  • endocytosis could also cause alteration in antibody metabolism, and accelerate the rate of antibody clearance in vivo.
  • the CLDN18.2 antibodies of the present disclosure did not cause significant cellular endocytosis and thus did not down-regulate the level of the target antigen CLDN18.2 on the cell surface, nor did they affect the antibody metabolism in vivo.
  • the defucosylated forms of the CLDN18.2 antibodies were further prepared. Specifically, the heavy and light chain coding sequences of the antibody were cloned into a mammalian GS expression vector capable of stable expression, with both the heavy and light chain coding sequences driven by the CMV promoter. The expression vector was transfected into CHO-K1 cells domesticated in serum-free and suspension culture and the domesticated CHO-K1 cell line with Fut8 gene knockout. Stable expression cell lines were screened and selected using MSX. After stable expression cell lines were obtained, they were cultured in shake flasks for 12-14 days, supplemented with supplemental medium in between as needed.
  • the culture supernatant was collected, and after filtering, the expressed antibodies were captured through a protein A chromatography column.
  • the antibodies were eluted and dialyzed with PBS to obtain purified antibodies.
  • the 18.2-A1, 18.2-A2, 18.2-A3, 18.2-A4, 18.2-A5 and 18.2-A6 antibodies expressed in the CHO-K1 cell line with Fut8 gene knockout were named 18.2-A1F, 18.2-A2F, 18.2-A3F, 18.2-A4F, 18.2-A5F and 18.2 A6F.
  • glycosylation modification of the defucosylated antibodies expressed in the CHO-K1 cell line with Fut8 gene knockout and the common antibodies produced in the CHO-K1 cells were analyzed.
  • the glycosyls of the antibodies were obtained by digesting 100 ⁇ g of the antibodies by trypsin and glycopeptidase followed by purification, and were subjected to fluorescence tandem mass spectrometry analysis.
  • the identification of various glycosyls relies on their mass-to-charge ratio m/z, and the percentage of glycosyl groups was calculated from the percentage of area tested by fluorescence.
  • glycosyl numbering 5 numbers respectively express the numbers of various sugars: six-carbon sugar (galactose, mannose, or glucose), N-acetylhexosamine (GlcNA or GalNAc), fucose (abbreviated as FUC), N-acetylneuraminic acid (Neu5Ac), and N-hydroxyacetylneuraminic acid (Neu5Gc).
  • the third number indicates the number of fucose. As shown in Table 4 below: Table 4.
  • Fc ⁇ RIIIa (Sino Biological Inc, 10389-H08C1) was diluted to 0.1 ⁇ g/ml in HBS-EP buffer as a ligand, and 18.2-A1F and 18.2-A1 antibody samples were diluted to 360 ⁇ g/ml, 120 ⁇ g/ml, 40 ⁇ g/ml, 13.3 ⁇ g/ml and 4.4 ⁇ g/ml respectively as analytes.
  • the ligand Fc ⁇ RIIIa was immobilized using an indirect capture method.
  • the test for each sample includes three Start up, one zero concentration control, five gradient concentration samples and one replicate sample (reference).
  • the chip was regenerated with 10 mM glycine-HCl, pH 1.5 regeneration solution after each cycle.
  • Each concentration cycle of the analyte was set with a capture time of 60s and a flow rate of the ligand solution of 10 ⁇ l/min; a binding time of ligand and analyte of 180s, and a flow rate of the analyte solution of 30 ⁇ l/min; and a dissociation time of 180s.
  • the CM5 chip coupled with Anti-His IgG was placed in the slot for detection and analysis.
  • the raw data were imported into the BIACORETM XI00 analysis software with the value of zero concentration control subtracted, and the value of reference channel was subtracted to eliminate the volume effect, and the affinity analysis method was used to fit the graph in steady-state mode and sort the data.
  • the Fc ⁇ RIIIa receptor is able to activate the NF-AT transcription factor pathway in effector cells upon binding to the FC region of the antibody.
  • detection of the NF-AT-mediated reporter gene intensity can reflect the intensity of Fc ⁇ RIIIa receptor activation.
  • a promoter containing the NF-AT binding site was inserted in a luciferase reporter gene expression vector and stably transfected into a Jurkat cell line.
  • high FC binding Fc ⁇ RIIIa V158 was also stably transfected into Jurkat cells, thereby constructing a functional cell line that can sense the intensity of Fc ⁇ RIIIa receptor activation (Fc ⁇ RIIIa-Jurkat).
  • SNU601 gastric cancer cells stably expressing CLDN18.2 (SNU601-CLDN18.2) were taken, diluted to 4 ⁇ 10 5 /ml, and mixed with Fc ⁇ RIIIa-Jurkat cells in a ratio of 1:6. 100 ⁇ l of the mixed cells were added to a 96-well plate, followed by the addition of fold-diluted antibodies, respectively, and incubated statically at 37°C for 6h. Subsequently, the luciferase activity was tested with the One-Glo kit (promega).
  • Combined chemotherapy regimen including EOF is the main available treatment method for gastric cancer.
  • EOF-sensitive cell lines undergo varying degrees of cell division cycle blockade, proliferation inhibition and apoptosis after EOF treatment.
  • KATOIII cells are a human gastric epithelial cancer cell line that expresses a very low level of CLDN18.2, with only about 5.2% of cells showing significant positivity in flow cytometry ( Figure 11 ). Due to the low level of CLDN18.2 expression, in the antibody-induced Fc ⁇ RIIIa receptor activation experiment with the KATOIII cells and Fc ⁇ RIIIa-Jurkat cells co-incubated (experimental method as described in Example 13), the 18.2-A1F and 18.2-A6F antibodies did not cause Fc ⁇ RIIIa receptor activation ( Figure 12A ).
  • KATOIII cells were treated with sublethal doses of EOF (Epirubicin: 300 nM; Oxaliplatin: 130 nM; 5-fluorouracil: 561.3 nM) for 48h, microscopic observation and flow cytometry revealed an increase in cell volume, rounding and a decrease of cells at division phase. It was indicated that the cells stayed in the division phase. However, the cells remained alive and did not show obvious apoptosis (results not shown).
  • the EOF-treated KATOIII cells were co-incubated with Fc ⁇ RIIIa-Jurkat cells and CLDN18.2 antibodies of different concentrations. The results showed that the 18.2-A1F and 18.2-A6F antibodies can induce significant Fc ⁇ RIIIa receptor activation as compared to the isotype control (ISO) ( Figure 12B ).
  • the currently commonly used chemotherapeutic drug EOF had an inhibitory effect on the growth of gastric cancer cells. Following the treatment with EOF, it enhanced the CLDN18.2 antibody-induced Fc ⁇ RIIIa receptor activation. Without wishing to be bound by theory, the effect may be due to the treatment with the chemotherapeutic drug promoting the ADCC effect of CLDN18.2 antibodies through up-regulation of antigen expression on cells and other mechanisms.
  • PBMC Peripheral blood leukocytes
  • donor 1 and donor 2 Peripheral blood leukocytes
  • SNU601 gastric cancer cells stably expressing CLDN18.2 SNU601-CLDN18.2
  • PBMC peripheral blood leukocytes
  • PBMC peripheral blood leukocytes
  • 100 ⁇ l of the mixed cells were added to a 96-well plate, and then the fold-diluted antibodies were added separately and incubated statically at 37°C for 24 hours. Subsequently, cell viability was tested using the lactate dehydrogenase (LDH) assay (promega), which determines the absorbance at OD490 by microplate reader.
  • LDH lactate dehydrogenase
  • HELA cells stably expressing CLDN18.2 were taken and resuspended in medium containing 1% serum with inactivated complement, and were counted, and the cell concentration was adjusted to 2 ⁇ 10 5 /ml with cell viability greater than 90%. 50 ⁇ l of cells per well were added in a 96-well plate, followed by the addition of fold-diluted antibodies respectively and 50 ⁇ l of diluted human serum. The cells were incubated at 37°C for 2 hours. Subsequently, the cell lysis rate was measured by the CCK8 method. The cell lysis rate was calculated as follows:
  • Example 17 In vivo tumor killing activity of CLDN18.2 antibody
  • PBS blank control
  • the 18.2-A1F antibody treatment groups at the doses of 5 mg/kg and 10 mg/kg showed good tumor suppression effects as compared to the PBS group and the IgG isotype control antibody group, and there was no obvious effect difference between the 5 mg/kg and 10 mg/kg groups.
  • Example 18 In vivo tumor killing activity of the combination of CLDN18.2 antibody and chemotherapeutic drug
  • KATOIII expressed a very low level of CLDN18.2 when cultured in vitro in IMDM medium, with only about 5.2% of the cells showing obvious positivity in flow cytometry ( Figure 11 ).
  • KATOIII cells with high CLDN18.2 expression were selected to inoculate NPG immunodeficient mice for xenograft model experiments ( Figure 16 ).
  • ISO isotype control antibody
  • EOF Epirubicin: 1 mg/kg
  • Oxaliplatin 3 mg/kg
  • 5-fluorouracil 30 mg
  • 18.2-A1F is capble of effectively inhibiting tumor growth as compared to the EOF group and the control antibody group, and the combination of 18.2-A1F with EOF further significantly reduced tumor growth, which indicated the potential of the combination of 18.2-A1F with chemotherapy in the clinical treatment of cancers.

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